High Resolution Structural Biology of the SRP GTPase

描述(由申请人提供)：拟议研究的长期目标是对两种GTP酶的分子功能机制有一个准确的了解，这两种酶在信号识别颗粒(SRP)介导的靶向分泌性和膜性蛋白中起核心作用。这两个蛋白质，SRP的信号序列识别亚单位FFH和它的膜相关受体FtsY，在翻译的核糖体新生链复合体从细胞质SRP转移到膜转位蛋白的过程中经历了分子握手。值得注意的是，这两种蛋白质的GTP酶结构域是结构上的同源物，并且直接相互作用。这种瞬时蛋白：蛋白质复合体的形成机制是理解根本重要的SRP靶向机制的核心。在这里，提出了三条解决其结构基础的研究路线：第一，正在-~1.0A分辨率下确定脱氧核糖核酸结合的FTH GTP酶‘NG’结构域的结构。已完成的结构应允许详细和准确地分析在较低分辨率下确定的结构中未显示的重要功能结构元素。其次，FFH和FtsY的NG结构域的稳定络合物已经被捕获在非水解性GTP类似物的存在下，并将进行生化表征，目的是结晶该络合物并确定其X射线结构。已经获得了小的“铅”晶体。FfhIFtsY NG复合体的结构将是理解SRP与其受体相互作用的分子细节的基础。最后，将进行生化和定点突变研究，以测试解决两种蛋白质之间相互作用的结构基础的特定假设。这项工作将以前两个目标的成果为基础。未来的研究将致力于了解为什么在SRP靶向途径的后续步骤中会出现两个同源的‘NG’GTP酶。由于SRP GTP酶是一组知之甚少的GTP酶的成员，表现出不同于经典的GTP酶开关的功能逻辑，因此了解靶向复合体形成的结构基础不仅对于SRP来说是重要的，而且对于理解“组装激活的”GTP酶如何建立在共同的GTP酶折叠上以利用GTP结合和水解来组织细胞成分发挥功能也是重要的。

DESCRIPTION (provided by applicant): The long term objective of the proposed research is to obtain a precise functional understanding of the molecular mechanisms of the two GTPases that play a central role in signal recognition particle (SRP) mediated targeting of secreted and membrane proteins. The two proteins, Ffh, the signal sequence recognition subunit of the SRP, and FtsY, its membrane-associated receptor, undergo a molecular 'handshake' during transfer of the translating ribosome nascent chain complex from the cytosolic SRP to the membrane translocon. Remarkably, the GTPase domains of the two proteins are structural homologs and interact directly. The mechanism for formation of this transient protein:protein complex is central to understanding the fundamentally important SRP targeting mechanism. Here, three lines of investigation to address its structural basis are proposed: First, the structures of the apo- and nucleotide-bound Fth GTPase 'NG' domain are being determined at -~ 1.0 A resolution. The completed structures should allow detailed and accurate analysis of functionally important structural elements that are not revealed in structures determined at lower resolution. Second, the stable complex of the NG domains of Ffh and FtsY has been trapped in the presence of a non-hydrolyzable GTP analog and will be characterized biochemically with the aim of crystallizing the complex and determining its X-ray structure. Small 'lead' crystals have been obtained. The structure of the FfhIFtsY NG complex will be fundamental for understanding the molecular details of the interaction of SRP with its receptor. Finally, biochemical and site-directed mutagenesis studies will be carried out to test specific hypotheses that address the structural basis of the interaction between the two proteins. This work will build on the results of the two previous aims. Future studies will be directed towards understanding why two homologous 'NG' GTPases occur at subsequent steps in the SRP targeting pathway. Because the SRP GTPases are members of a poorly understood group of GTPases that exhibit a functional logic different from the classic 'GTPase switch', an understanding of the structural basis for formation of the targeting complex will be of importance not only with respect to the SRP, but also to understanding how 'assembly-activated' GTPases build on the common GTPase fold to harness GTP binding and hydrolysis to organize cellular components for function.